ORIGINAL ARTICLE

Diversity of plasmids and Tn1546-type transposonsamong VanA Enterococcus faecium in Poland

E.Wardal1 & A. Kuch2& I. Gawryszewska1 & D. Żabicka2 & W. Hryniewicz2 & E. Sadowy1

Received: 21 June 2016 /Accepted: 26 September 2016 /Published online: 17 October 2016# The Author(s) 2016. This article is published with open access at Springerlink.com

Abstract The objective of this study was to investigate theantimicrobial resistance, Tn1546 transposon variability andplasmid diversity among Polish vancomycin-resistantEnterococcus faecium (VREfm) isolates of VanA phenotypein the context of their clonal structure. Two hundred sixteenclinical VREfm isolates collected between 1997 and 2010were studied by antimicrobial susceptibility testing, MLST,MLVA and detection of IS16, espEfm, pilA, intA andplasmid-specific genes by PCR. Tn1546 structure was re-vealed by overlapping PCR and sequencing. Selected isolateswere subjected to PFGE-S1 and Southern hybridization anal-yses. The vast majority of the isolates (95.8 %) belonged tolineages 17/18 (during the whole study period 1997–2010)and 78 (mostly in 2006–2010) of hospital-adapted merocloneof E. faecium. All isolates displayed a multi-drug resistancephenotype. Twenty-eight Tn1546 types (including 26 novelones) were associated with eight different ISs (IS1216,IS1251, ISEfa4, ISEfa5, ISEfm2, ISEf1, IS3-like, ISEfm1-like). The vanA-determinant was typically located on plas-mids, which most commonly carried rep2pRE25, rep17pRUM,rep18pEF418, rep1pIP501, ω-ε-ζ and axe-txe genes. VanA iso-lates from 1997–2005 to 2006–2010 differed in clonal com-position, prevalence of gentamicin- and tetracycline-

resistance and plasmidome. Our analysis revealed high com-plexity of Tn1546-type transposons and vanA-plasmids, andsuggested that diverse genetic events, such as conjugationtransfer, recombination, chromosomal integration and DNAmutations shaped the structure of these elements amongPolish VREfm.

Introduction

In the past 20 years, vancomycin-resistant enterococci(VRE) have emerged as nosocomial pathogens world-wide. In Poland, the first VRE outbreak due toEnterococcus faecium (VREfm) of VanA phenotypestarted in December 1996 in the Gdańsk MedicalUniversity [1]. The vast majority of VREfm observedworldwide belongs to a specific hospital meroclone, ini-tially described as clonal complex 17 (CC17), later di-vided into three distinct lineages 17, 18 and 78 basedon multilocus sequence typing (MLST) analyses [2, 3].Recently, the approach called Bayesian Analysis ofPopulation Structure (BAPS), applied to the E. faeciumMLST data delimited two groups within the hospitalmeroclone, 2–1 and 3–3, corresponding to lineages 78and 17/18, respectively [4]. Strains belonging to thehospital meroclone are ciprofloxacin- and ampicillin-re-sistant, enriched in putative virulence traits, and show adistinct genetic repertoire, including cell surface proteingenes (fms), regulatory genes, putative pathogenicityislands, plasmids, insertion sequences (IS) and integrat-ed phages, which promote their adaptation [5–7]. Thepresence of IS16 and the E. faecium-specific esp gene(espEfm), carried on the integrative conjugative elementICEEfm1, together with the intA integrase gene, are

Electronic supplementary material The online version of this article(doi:10.1007/s10096-016-2804-8) contains supplementary material,which is available to authorized users.

* E. Sadowyewasadowy@cls.edu.pl

1 Department of Molecular Microbiology, National MedicinesInstitute, Chełmska 30/34, 00-725 Warsaw, Poland

2 Department of Epidemiology and Clinical Microbiology, NationalMedicines Institute, Chełmska 30/34, 00-725 Warsaw, Poland

Eur J Clin Microbiol Infect Dis (2017) 36:313–328DOI 10.1007/s10096-016-2804-8

proven molecular markers of hospital-associatedE. faecium [8–10].

Several glycopeptide-resistance phenotypes have been de-scribed so far, with VanA and VanB being the most commonin enterococci isolated from hospital infections [11]. The vanAgene cluster is carried on Tn1546-type transposons [12],which show a significant degree of heterogeneity, associatedwith presence of point mutations, deletions and presence ofvarious ISs [13, 14]. A few studies demonstrated the locationof Tn1546 on Inc18, pRUM-like, pMG1-like, and pLG1 plas-mids [15, 16]; however, the knowledge of vanA-plasmids andtheir epidemiology is still far from being satisfactory and maydiffer significantly among countries.

In Poland, hospital VRE isolates are continuously submit-ted for confirmation and further analyses to the NationalReference Centre for Susceptibility Testing (NRCST), locatedat the National Medicines Institute in Warsaw. The aim of thisstudy was to characterize E. faecium VanA isolates collectedby the NRCST since 1997 until the end of 2010, focusing onthe Tn1546 transposon variability and vanA-plasmid diversityin the context of the clonal structure of VREfm isolates toprovide the country-wide picture of these important hospitalpathogens.

Materials and methods

Bacterial isolates and susceptibility testing

The study comprised 216 consecutive, non-repetitive (1isolate per patient) VREfm VanA isolates received bythe NRCST from 42 hospitals in 24 cities in Polandover the period 1997–2010. Part of the isolates analyzedin this work correspond to strains partially tested inprevious surveillance studies, including: 108 VanA rep-resentatives of the VREfm collection from 1997 to 2005[17] and 20 representative isolates of a E. faeciumVanA outbreak in 2009 [18]. The majority of isolates(n = 137) were derived from 11 VanA outbreaks and theremaining 79 isolates were reported as single isolations.Of the 216 isolates, 211 (97.7 %) were from hospital-ized patients and five (2.3 %) were from the hospitalenvironment. Among the isolates from hospitalized pa-tients, a total of 37 isolates (17.5 %) were from inva-sive infections (31 isolates from blood and 6 from othersources); 52 isolates (24.6 %) were from non-invasiveinfections (21 from urine, 18 from wounds, and 13 fromother sources) and 122 (57.8 %) represented faecal car-riage. Antimicrobial susceptibility of 88 isolates, notinvestigated previously, was tested by the Etest method(bioMérieux, Marcy l’Etoile, France) for daptomycin,t e i cop l an in and vancomyc in and by a b ro thmicrodilution method [19] for the remaining compounds

(Table 1). Multidrug-resistant (MDR) isolates were de-fined as recommended [20]. Vancomycin-resistance de-terminants were detected by PCR as described previous-ly [21] with the E. faecium BM4147 and E. faecalisV583 strains as positive vanA and vanB controls,respectively.

DNA isolation and genotyping of isolates

Total DNA of isolates was extracted using Genomic DNAPrep Plus kit (A&A Biotechnology, Gdansk, Poland).Multilocus VNTR analysis (MLVA), MLST, and detectionof 19 rep families and the unique reppMG1 gene were per-formed as described [22–24]. Sequence types (STs) weregrouped to CCs by the comparative eBURST analysis per-formed against the whole E. faecium MLST database. PCRdetection of IS16, espEfm, fms21 (pilA), reppLG1, plasmid ad-diction systems, relaxase genes, and intAICEEfm1 was per-formed as described (Supplementary Table 1 and referencestherein). DNA of enterococcal isolates from our laboratorycollection [17, 18, 25] served as positive controls.

Plasmid profiling, hybridization analyses, Tn1546 typingand statistical analysis

DNA in agarose plugs was obtained as described [21], treatedwith S1 nuclease (Takara Bio, Japan) and separated by PFGEwith Lambda Ladder PFG marker (New England Biolabs,Beverly, MA) [26] followed by blotting onto the Hybondmembrane (GE Healthcare, Buckinghamshire, UK) by capil-lary transfer. Hybridization was carried out using theAmersham ECL Random-Prime Labelling and DetectionSystem (GE Healthcare, Buckinghamshire, UK). Tn1546transposon was investigated by PCRmapping and sequencing(Supplementary Table 1 and references therein) of selectedregions encompassing 7571 bp out of 10851 bp (i.e., ∼70 %of the transposon, Fig. 1). The Tn1546 sequence of E. faeciumBM4147 (GenBank acc. no.: M97297) [12] was used as areference. The nomenclature of Tn1546-type transposons inthe present study was based on the following alphanumericcode: the ‘A’ types (A1-A6) referred to transposon variants ofthe wild type (wt) Tn1546 structure (A1) not interrupted byinsertion sequences; the ‘B’ types contained 1–3 copies ofIS1216 (B, BB, BBB types); the C, D, E, F, G, H and I typescarried IS1251, ISEfa5, ISEfa4, ISEfm2, ISEf1, ISEfm1-likeand IS3-like elements, respectively. Transposons with morethan one IS type were described by a two-, three- or four-letter code (e.g., ‘BC’ with both IS1216 and IS1251). TheArabic numerals indicated differences in the presence of par-ticular point mutations as well as the orientation of ISs and thelocalization of their insertion sites (e.g., B1-B4). The novelISEfm1-like sequence was submitted to GenBank

314 Eur J Clin Microbiol Infect Dis (2017) 36:313–328

(KT719407). Chi-square test was used to assess the differ-ences of distributions, with p ≤ 0.05 considered significant.

Results

Susceptibility to antimicrobial agents

All isolates were resistant to vancomycin and teicoplanin(Table 1) and carried vanA. Resistance to ampicillin, cipro-floxacin, tetracycline, chloramphenicol, gentamicin and strep-tomycin (high level) was prevalent or highly prevalent and allisolates showed the MDR phenotype. A significant decline inthe prevalence of both tetracycline-resistance (from 68.9 to52.3 %, p = 0.01) and high-level gentamicin resistance (from92.1 to 64.4 %, p < 0.0001) was found between the 1995–2005 and 2006–2010 periods. A single isolate was resistantto linezolid and two isolates to quinupristin/dalfopristin. Allisolates were susceptible to tigecycline and daptomycin.

MLVA, MLST, IS16 and virulence markers detection

MLVAwas performed for 196 isolates and these results wereanalysed together with data obtained earlier for 20 isolates fromthe 2009 outbreak [18]. Among 216 isolates, 37 different

MLVA types (MTs) and three incomplete profiles (due to lackof VNTR7 amplification) were observed, that included 207 andnine isolates, respectively (Supplementary Table 2). MT1,MT159, MT25 and MT13 were most prevalent, with 36, 34,26 and 20 isolates, respectively. All MT159 isolates except onewere isolated in 2006–2010 (p ≤ 0.0001), in contrast to isolatesof MT25, which all except one were isolated in 1997–2005(p = 0.0001). The MT1 isolates showed a similar frequencyover the whole study period (13.2 % vs 21.6 %, p = 0.07). Inthe present study, STs and the presence of IS16, espEfm, intAand pilAwere determined for 88 isolates not encompassed bythe previous studies (i.e., 68 isolates from 2006 to 2010 and 20isolates from 2001 to 2002), and these results were analysedwith the data obtained previously for the remaining 128 iso-lates. Altogether, 40 different STs were found, with 18 STs(45.0 %) represented by single isolates. The vast majority ofthe STs, i.e. 36 STs representing 207 isolates (95.8 %),belonged to the hospital-adapted meroclone of E. faecium; lin-eage 17/18 included 31 STs with 165 isolates and was presentduring the whole period 1997–2010, while lineage 78 includedfive STs characteristic for 42 isolates, occurring mostly in2006–2010 (p ≤ 0.0001). IS16 was present in 207 (95.8 %)isolates and 186 (86.1 %) isolates harboured the espEfm gene.The integrase gene intA and the pilA gene were found in 181(83.8 %) and 214 (99.1 %) isolates, respectively.

Table 1 MIC values for E. faecium VanA isolated in Poland during the period 1997–2010

Compound/phenotype 1997–2010N = 216

1997–2005N = 128

2006–2010N = 88

MIC breakpoints/ECOFF (μg/ml)

Number (%)non-susceptible

Number (%)non-susceptible

MIC 50

(mg/l)MIC90

(mg/l)Number (%)non-susceptible

MIC 50

(mg/l)MIC90

(mg/l)S ≤ R >

Vancomycina 216 (100) 128 (100) 512 >512 88 (100) >256 >256 4 4

Teicoplanina 216 (100) 128 (100) 64 128 88 (100) 48 >256 2 2

Ampicillina 215 (99.5) 128 (100) >128 >256 87 (98.8) 128 >256 4 8

HLGRa 172 (79.6) 118 (92.2) >1024 >1024 54 (64.4) >1024 >1024 128 128

HLSRa 167 (77.3) 112 (87.5) >1024 >2048 55 (62.5) >1024 >2048 512 512

HLAR 28 147 (68.1) 110 (86) >1024 >1024 37 (42) >1024 >2048 - -

Quinupristin/dalfopristina

2 (0.9) 1 (0.8) 1 2 1 (1.1) 1 1.5 1 4

Linezolida 1 (0.5) 0 (0) 1 4 1 (1.1) 1 2 4 4

Tigecyklinea 0 (0) 0 (0) 0.06 0.19 0 (0) 0.06 0.25 0.25 0.5

Tetracyclineb 135 (62.2) 89 (68.9) 64 128 46 (52.3) 8 128 4 4

Ciprofloxacinb 215 (99.5) 127 (99.2) 128 >256 88 (100) 128 256 4 4

Daptomycinb 0 (0) 0 (0) 2 3 0 (0) 2 3 4 4

Chloramphenicolc 53 (24.4) 30 (23.2) 8 16 23(26.1) 8 16 8 ≥32c

MDR28 216 (100) 128 (100) nc nc 88 (100) nc nc – –

nc not calculated, n number of isolates

The results were interpreted following the European Committee on Antimicrobial Susceptibility Testing (EUCAST)-approved breakpoints [53] and theEcological Cut-Off (ECOFF) values for compounds without defined breakpoints (http://mic.eucast.org/Eucast2/, last accessed 20th July 2015). Forchloramphenicol the Clinical and Laboratory Standards Institute (CLSI) breakpoints were used [19]a Interpretation according to the EUCAST clinicalbreakpoint valueb Interpretation according to the EUCAST Ecological Cut-off (ECOFF) valuec Interpretation according to CLSI breakpoint value

Eur J Clin Microbiol Infect Dis (2017) 36:313–328 315

Tn1546-1 Orf1-5 Orf2-F Orf2-R

Orf2-F2

vanR-R

vanS vanH2

vanS1

vanA1 vanX2vanX1 vanX-F

vanY1 vanY2-R

vanZ1

vanZ2

orf1 orf2 vanR vanS vanH vanA vanX vanY vanZ

8138

A2

9063

A3

A57747

A4Δ 5896-5939

IS1216 DRs:ATTGGTGA

8907-8900

B1

IS1216RDRs:GGCAGAGC

8746.-8739.7747

B3

9063

A61915 9063

IS1216V

3902

B2nd.

A1

1 2 3 4 5 6 7 8 9 10 10,851 kb

Fig. 1 Diversity of Tn1546 transposon types among E. faecium VanAisolates. Position of primers used in PCR mapping and sequencingindicated by arrows with primer names; open rectangles, transposongenes; stars, positions of point mutations; analyzed areas of the

transposon shadowed; dashed lines, deletions in the left arm of thetransposon; filled rectangles, deletions within the transposon; verticalarrow, triangles with arrows, the IS positions; single-nucleotide insertionin vanY

316 Eur J Clin Microbiol Infect Dis (2017) 36:313–328

Structural diversity of Tn1546 transposons

The structure of Tn1546-type transposons was determinedfor 187 isolates while for 20 isolates the structure of thetransposon (representing types A1 and G, Fig. 1) had beenpublished earlier [18]. In the case of nine isolates the

structure of the transposon could not be determined inspite of repeated attempts due to lack of PCR productsfor some parts of the transposon, which might have beencaused by sequence polymorphism(s) within PCR primersannealing sites, and discrepancies of sequencing results incertain regions, likely associated with the presence of

1915

IS1251DRs:ATAATTTT

5820.-5813. 7658 8234 9692

C2

1915

IS1251DRs:ATAATTTT

5820.-5813 8234 9692

C1

IS1216V

B4nd. 3902.

T in pos. 9063-9064

ISEfa5DRs:GAAATATT

9013.-9006.D

ISEfa4DRs: none

8631.-8633. 9063 E

ISEfm2DRs:GACTGAAA

9051.-9044.F

ISEf1DRs:GACTGAAA

9051.-9044.G

7747 Fig. 1 continued.

Eur J Clin Microbiol Infect Dis (2017) 36:313–328 317

more than one transposon in a single isolate. Twenty-eighttransposon types, including 26 new ones, were discernedin the analyzed group (Fig. 1). The most predominanttypes, including C1 (40 isolates), B2 (n = 38), A3 (n =36), G (n = 25), E (n = 14), A1 (n = 13) and D (n = 7),were associated with several STs and typically showed amulticenter distribution. Eight different ISs were detectedwithin Tn1546, including IS1216, IS1251, ISEfa4,ISEfa5, ISEfm2, ISEf1, IS3-like and ISEfm1-like. Themost common IS1216 was present in all 16 B-type trans-posons, both in the direct and reversed orientations, withfive different types of 8-bp direct repeats. These B-type

transposons were found in Gdańsk and Warsaw, as well asin 14 other cities. IS1251 was associated with sevenTn1546 types (C1-C2, BC1-BC5) and present in 53 iso-lates (25.6 %), which mainly originated from Kraków andWarsaw. In these isolates, IS1251 was always inserted inthe vanS-vanH intergenic region of the transposon at theposition 5813. The D, E, F and G types of transposonwere characterized by the presence of ISEfa5, ISEfa4,ISEfm2 and ISEf1 in the vanX-vanY intergenic region,respectively. These types were generally limited to oneor two centres, with the exception of the G type, whichapart from the outbreak in two Warsaw hospitals [18]

8234

IS1216

3324.

IS1216

5603 Δ 8909-8912

BB1

1915

IS1251DRs:ATAATTTT

5820.-5813. 8234 7658

IS1216R

Δ 8734-9018BC4

1915

IS1251DRs:ATAATTTT

5820.-5813. 8234 7658

IS1216R

BC1DRs:ATTGAAGA

8726.-8733.

IS1216R

3344.

IS1251DRs:ATAATTTT

5820.-5813. 7658 8234 9692

BC2

1915

IS1251DRs:ATAATTTT

5820.-5813. 8234 7658

IS1216R

Δ 8634-8725BC3

8234

IS1216

3916.

IS1216

5603 Δ 8909-8912

BB2

Fig. 1 continued.

318 Eur J Clin Microbiol Infect Dis (2017) 36:313–328

occurred in seven other centres. An insertion of an IS in98 % identical to ISEfm1 (GenBank no. AF138282) in thevanX-vanY intergenic region resulted in the BH-type oftransposon.

The variability of Tn1546 was additionally associatedwith the presence of deletions, insertions and point mu-tations. In the A4 type, a 44 nt deletion in the vanS-vanH intergenic region (nt 5896–5939) was observed.Other deletions, located in orf2, vanX and the vanX-vanY intergenic region coincided with the presence ofISs (six types: BB1, BB2, BC4, BC5, BBI, BBBI2).Seven different point mutations were detected, including

four known previously (T7658C, G7747T, G8234T,C9692T) [18, 27, 28] and three new ones (G5603A,A8138G and G9063T). The G5603A mutation resultedin the A80T change in VanS and the G9063T mutationin the L4F change in VanY. The B4 type, found in twoindependent isolates, demonstrated the presence of anovel single-nucleotide T insertion between nt 9063–9064 within the vanY gene, resulting in translationalframeshift and a truncated VanY. Nevertheless, thesetwo isolates showed high MIC values for vancomycin(>512 mg/L in both cases) and teicoplanin (64 and>128 mg/L).

8234 3331.-3324.

IS1216 DRs:GTAAATCC

8920.-8913.

DRs:TCAAAAAGIS1216

120.

IS3-like

BBBI1

305 bp IS1216

IS1216

3902.

ISEfm1-likeDRs:AGTAGATA

8723.-8716.

BHnd.

8234

IS1216 DRs:GTAAATCC

nd -8913.

IS1216

120.

IS3-like

BBBI2

305 bp IS1216

Δ 3332-3917

120.

IS1216R

Δ 8659-8874

BBI

IS1216 IS3-like

Δ 974-1877

IS1216 IS3-like

BI

Δ 974-1877

120.3998. 6062.

3998. 6062.

9213. 10258.

9213. 10258.

IS1216

BC5Δ 8659.-8725.

8234 1915 7658

IS1251DRs:ATAATTTT

5820.-5813.

Fig. 1 continued.

Eur J Clin Microbiol Infect Dis (2017) 36:313–328 319

Plasmid gene content among VREfm and diversityof vanA-plasmids

PCR-based typing of plasmid replication initiator genes (rep)was performed for 196 isolates and these results were com-bined with the data for 20 isolates, published previously [18].Altogether, ten rep-types were observed among VREfm-VanA(Fig. 2). Isolates carried from one up to seven different repgenes, with 4.7 rep genes per isolate on average. Isolates pos-itive for rep1pIP501, rep7pT181 and reppMG1 appeared mainly inthe period 1997–2005, while rep11pEF1071 gene was typicalfor isolates obtained in 2006–2010. The plasmid stabilizationsystems axe-txe and rep17pRUM were present in the majorityof isolates. Another system, ω-ε-ζ, was also quite common,predominantly among rep2pRE25-carrying strains, and oc-curred mainly in the period of 2006–2010. Two additionalsystems, mazEF and relBE, were observed only in 2003 forsix and two isolates, respectively. The relpCIZ2 and relpEF1relaxase genes were prevalent, and additionally relpHTß andrelpRE25 were detected. The majority of relpRE25-positive iso-lates (n = 22, 91.7 %) were also rep2pRE25-positive, however,most of 169 rep2pRE25-positive isolates lacked this relaxase.Isolates carrying relpHTß dominated in 1997–2005 (40 out of45 relpHTß-positive isolates) and the majority of relpHTß -positive isolates also harboured reppMG1 (n = 43, 95.5 %).

Fifty-two isolates, obtained from 24 medical centres overthe whole study period and representing 26 different STs and21 Tn1546 types were selected for PFGE of S1-digested DNAand hybridization analyses. Additionally, the results obtainedpreviously for three isolates from the 2009 outbreak [18] wereincluded for comparative purposes. Investigated isolates

showed the presence from one up to 11 plasmid bands perisolate in PFGE-S1 analyses. Subsequent hybridization withthe vanA probe revealed the presence of 86 vanA-plasmidswith up to four such plasmids in an isolate, and two cases ofchromosomal localization of vanA (Table 2). Further hybridi-zation studies showed the co-localization of vanA determi-nants with all six tested rep types, including rep2pRE25,rep17pRUM, rep18pEF418, rep1pIP501, reppLG1 and reppMG1 thataccounted for 40.7 % (n = 35), 40.7 % (n = 35), 24.4 % (n =21), 19.8 % (n = 17), 5.8 % (n = 5) and 1.2 % (n = 1) of vanA-plasmids, respectively. The vanA-plasmids with rep1pIP501were limited to isolates from 1997 to 2005, circulating intwo hospitals in Poznań. These plasmids differed by size(from ca. 30 to ca. 265 kb) and presence of other rep andtoxin-antitoxin genes, and carried four different types ofTn1546, with A3 being predominant (8 out of 13 isolatesharbouring vanA-plasmids with rep1p IP501) . ThevanA-plasmids with rep2pRE25, rep17pRUM and rep18pEF418genes showed a multicentre distribution and occurred duringthe whole study period. In total, 37 (43 %) vanA-plasmidswere associated with more than a single rep type and 21vanA-plasmids (24.4 %), present in 11 isolates, did not hybrid-ize with any of the tested rep genes. With a single exception,these latter plasmids were obtained during 2006–2010 (p =0.001). Five of the isolates with these unknown replicons con-comitantly carried three vanA-plasmids, ca. 30, 160 and380 kb in size, which did not hybridize with any probes oftoxin-antitoxin and relaxase genes tested. All these isolatescarried B2 transposons, but belonged to diverse STs andMTs, and originated from four different medical centres over2006–2010. Two toxin-antitoxin systems, ω-ε-ζ and axe-txe

% o

f is

ola

tes

70

117

43

1 1

7479

47

84

108

125

11

26

22

125 126

40

1

49

7

0

42

61 63

34

3

68

61

37

00 2

75 73

3

71

166

50

1

43

135 142

81 87

176 186

48

2 6

24

200 199

43

Fig. 2 Plasmid-associated gene distribution among Polish VREfm VanA. Number of isolates with a particular gene given above the graph bars

320 Eur J Clin Microbiol Infect Dis (2017) 36:313–328

were commonly carried by vanA-plasmids (35 and 32 plas-mids, respectively). The ω-ε-ζ system was characteristic forrep2pRE25 plasmids and axe-txe for rep17pRUM plasmids (71.4and 62.9 % of the respective vanA-plasmids). The gene spec-ifying pEF1-relaxase was located on 11 vanA-plasmids(12.8 %), with various rep types. Some of the presumed ge-netic events, that could be inferred on the basis of these anal-yses, include examples of transposon evolution within an en-terococcal strain, Tn1546 transposition among plasmids,conjugative transfer of plasmids, and their changes such asrecombination or chromosomal integration as proposed inTable 2.

Discussion

Currently, VREfm play an increasingly important role in nos-ocomial infections and are considered alert pathogens [29],with vanA as a main determinant of this phenotype withinmany countries [30]. In Poland VRE remain less prevalentthan in the United States or some European countries, e.g.our recent study revealed 7 % vancomycin-resistance amonginvasive E. faecium collected during 2010–2011 [31].Although we observed an increasing prevalence of VanBE. faecium [17], VanA is still most frequent among PolishVREfm ([31] and NRCST unpublished observations). In thepresent study we aimed at the characterization of clonality ofVanA-VREfm and genetic elements associated with this resis-tance determinant. Numerous reports show that in the case ofhuman nosocomial infections vancomycin resistance is almostexclusively acquired by the hospital-adapted meroclone ofE. faecium, now widespread all over the world [2, 3] andprevalent among invasive E. faecium in Polish hospitals[31]. In this study, the vanA determinant was carried by rep-resentatives of this meroclone with only a few exceptionslimited to the 1997–2005 period. These isolates might repre-sent intermediates, by which glycopeptides resistance deter-minants were introduced into hospitals. All isolates belongingto hospital meroclone, as expected, were resistant to both am-picillin and ciprofloxacin, and enriched in putative virulencetraits / markers such as IS16, espEfm, intAEfm and pili genes.The population structure determined for Polish VREfm VanAclosely resembled these of hospital-associated E. faecium inother countries. High diversity of STs/MTs is consistent withthe presence of polyclonal hospital population of E. faeciumthat subsequently acquires vancomycin resistance determi-nants [13, 14]. The vast majority of isolates grouped into hos-pital lineage 17/18, mostly represented by STs 17, 117, 18,132, 202 and lineage 78, which included STs 78, 192 and 412.In contrast to several other countries, where ST203 and ST16constituted a significant proportion of hospital E. faecium [3,14, 32, 33], in our population only one representative of ST16was found and ST203 was completely absent. The

characteristic change in the proportion of isolates belongingto lineage 17/18 and lineage 78 was observed since the year2005when lineage 78 started to be significantly more frequentin Poland. Our results are in agreement with observationsmade in other studies, suggesting waves of successfulE. faecium, first from lineage 17/18 and followed by lineage78 strains [4, 34]. This population shift, apparent inMLSTandMLVA, was additionally associated with a change inplasmidome composition and observed decreased resistancelevels to tetracycline and aminoglycosides.

Diversity of Tn1546 in VREfm is typical for this transpo-son, as reported by others [13, 14]. Nevertheless, in the presentstudy we observed several new variants of Tn1546. VanAtransposons indistinguishable from the Tn1546 A1 prototype[12] were frequently encountered in Europe, especially in thelate 1990s and 2000s [14, 27]. This type and its mutationalderivatives (A2-A6) were ubiquitous among early VREfm inour study. Single-nucleotide T insertion between nt 9063–9064 in the vanY gene of B4 type of transposon resulted in atranslational frameshift and a truncated translation product.This change, however, did not abolish the glycopeptide resis-tance. VanY is a membrane-associated D,D-carboxypeptidasethat hydrolyses the C-terminal D-Ala or D-Lac residue ofpeptidoglycan precursors but lacks transpeptidase activity.VanY, together with VanZ, represent accessory proteins,which are not required for the expression of glycopeptideresistance but increase its level [35]. Isolates with a deletionof vanY gene showed lower resistance levels to teicoplanin,likely due to the diminished transcription of vanZ while pointmutations in vanY, observed so far were not associated with aloss of protein function [27, 36].

Activity of various ISs represented a very importantfactor, contributing to the formation of several noveltransposon types. IS1216, the most common IS in ourstudy, characteristic for B-types, was detected at variouspositions of the transposon, and its insertion often resultedin deletions of adjacent sequences in ORF2, vanX and thevanX-vanY intergenic region, as observed by others [13,27]. The BI, BBI, BBBI types, apart from IS1216, exhib-ited the concomitant presence of a IS1216V-IS3-like ele-ment, originally reported in 1995 [37]. Since then, thiselement was described in several studies, which reportedintact as well as a 5′-truncated IS3-like sequence [27, 38],both of which were also detected in our study. The C-type, harbouring IS1251, was relatively frequent and theintegration sites of this IS were identical to those pub-lished by others [14, 37, 39]. Sequencing analysis alloweddiscerning the C1-type, specific for Krakow hospitals andthe C2-type, found in other cities. The D type transposon,containing ISEfa5, to our knowledge, represents the firstexample of this variant outside of South America [34].Type E represents the first insertion of ISEfa4 in thevanX-vanY intergenic region. This IS was described

Eur J Clin Microbiol Infect Dis (2017) 36:313–328 321

Tab

le2

Characteristicsof

theVanAplasmidom

eof

55selected

VREfm

isolates

StrainID

/year

ofisolation

Codeofmedicalcentrea

Tn1546

type

MLST

type

(lineage)

Num

berofV

anA

plasmid

bands

Hypotheticalgenetic

eventb

VanAplasmid

replicon

typesandstabilizatio

nsystem

s(approximatesize

inkb)c

1639/1997

Gd-a

BBBI1

407(17/18)

1rep17TA

1(45)

1641/1997

Gd-a

BBBI2

408(17/18)

1Chrom

osom

alintegrationof

50-kbplasmid

rep17rep18TA

1(50)

rep2

rep17rep18TA

1(chr)

3132/1998

Gd-p

A1

18(17/18)

2Transferof

40-kbplasmid

betweenST18

andST

411

strains,follo

wed

byplasmid

recombinatio

nor

transposition

ofA1

rep2

rep17TA

1(35)

rep2

TA2(40)

3136/1998

Gd-p

A1

411(singleton)

2rep2

TA2(40)

rep2

TA2(270)

7952/1999

Gd-p

nt381(17/18)

3rep2

rep17TA

1(35)

rep17TA

1(170)

rep17TA

1(320)

2509/2000

Po-1

A3

386(17/18)

2Chrom

osom

alintegration

of40-kbplasmid

rep2

(30)

rep2

TA1TA

2(40)

rep1

rep2

TA1

TA2(chr)

2524/2000

Po-1

A3

382(17/18)

2rep17TA

2(40)

rep1

TA2relpEF1

(140)

2712/2000

Po-1

A3

385(17/18)

3Transpositio

nof

A3or

plasmid

recombinatio

nin

ST385strain

TA2(40)

rep1

TA2(140)

rep1

rep p

LG1TA

2

relpEF1

(265)

1409/2002

Po-4

A3

385(17/18)

1rep2

rep p

MG1TA

1TA

2(250)

2506/2000

Po-1

A4

385(17/18)

2Derivativeof

A3in

ST385

strain,w

ithconcom

itant

change

ofplasmid

backbone

rep2

rep18TA

2(<30)

rep1

rep18TA

2(265)

291/2002

Po-2

A3

117(17/18)

1rep1

rep17TA

1TA

2(145)

1714/2003

Po-2

A3

117(17/18)

1Clonalspreadof

ST117with

265-kb

plasmid,followed

bytransposition

ofA3

amongplasmidsor

plasmid

recombinatio

n

rep1

(265)

710/2004

Po-2

A3

117(17/18)

4rep1

(265)

rep1

rep2

rep18TA

1TA

2(255,310,360)

914/2002

Po-2

A3

17(17/18)

1rep1

rep17TA

1(155)

2039/2003

Po-2

A3

410(17/18)

1rep1

(145)

3010/2003

Po-2

A3

192(78)

1rep1

rep2

rep18(145)

655/2007

Po-4

A6

549(78)

1rep2

TA1TA

2(40)

3003/2003

Po-2

E117(17/18)

1Transpositio

nof

Eor

plasmid

recombinatio

nin

ST117strain

rep1

rep2

rep18rep p

LG1

TA1(165)

2127/2004

Po-2

E117(17/18)

1rep1

rep p

LG1(165)

2512/2000

Po-1

D17(17/18)

1rep1

TA2(30)

1156/2002

Po-2

D382(17/18)

1rep1

rep2

TA2(50)

714/2003

Kr-1

C1

117(17/18)

1rep2

rep18rep17

rep p

LG1TA

1TA

2(450)

756/2003

Kr-1

C1

17(17/18)

1rep17rep18TA

1TA

2relpEF1(70)

1679/2003

Kr-1

C1

18(17/18)

1Clonalspreadof

ST18

strain

andconcom

itant

change

ofplasmid

size

bya

rep2

rep18TA

1TA

2relpEF1

(70)

3779/2004

Kr-4

C1

18(17/18)

1

322 Eur J Clin Microbiol Infect Dis (2017) 36:313–328

Tab

le2

(contin

ued)

StrainID

/year

ofisolation

Codeofmedicalcentrea

Tn1546

type

MLST

type

(lineage)

Num

berofV

anA

plasmid

bands

Hypotheticalgenetic

eventb

VanAplasmid

replicon

typesandstabilizatio

nsystem

s(approximatesize

inkb)c

presum

abledeletio

n(lossof

rel pEF1)

rep2

rep18TA

1TA

2(65)

4002/2005

Kr-3

C1

387(17/18)

1Plasmid

transfer

and∼1

5-kb

deletio

nrep2

rep18TA

1TA

2relpEF1

(55)

1332/2003

Kr-1

C1

132(17/18)

1Clonalspreadof

ST132strain

with

45-kbplasmid

harbouring

C1transposon

rep17TA

1(45)

1336/2003

Kr-1

C1

132(17/18)

1rep17TA

1(45)

2981/2003

Mi

ntd

132(17/18)

1Evolutio

nof

C1transposon

with

inthesameST132

strain

and45-kbplasmid

backbone

rep17TA

1(45)

2216/2005

Kr-1

C1

388(17/18)

1rep2

rep17rep18TA

1TA

2relpEF1(75)

84/2010

Gdy

C2

17(17/18)

1rep2

rep17TA

1TA

2(50)

3552/2009e

Wa-10

A1

18(17/18)

2AncestorforB2transposon,

associated

with

plasmidsof

unknow

nrep-type(s)

(<30)

(170)

3240/2006

Po-5

B2

17(17/18)

3Concomitant

transfer

ofthree∼3

0-,160-and380-kb

plasmidswith

unknow

nrep-type(s),carrying

B2

transposon,intodiverse

clonalbackgrounds

(30)

(160)

(380)

1930/2007

Wa-1

B2

64(17/18)

3(30)

(160)

(380)

4285/2008

Wa-1

B2

192(78)

3(30)

(160)

(380)

5151/2008

Osw

B2

18(17/18)

3(30)

(160)

(380)

1767/2010

Wa-3

B2

780(17/18)

3(<30)

(160)

(380)

5009/2009

Wa-2

B2

230(78)

3Transferof

160-kb

plasmid

ofunknow

nrep-type,follow-

ed bytransposition

ofB2

amongplasmidsor

plasmid

recombinatio

n

rep17TA

1(<30),

rep17(45)

(160)

3238/2006

SkB2

279(17/18)

2Transferof

30-kbplasmid

ofunknow

nrep-type,follow-

ed bytransposition

ofB2

amongplasmidsor

plasmid

recombinatio

n

(30)

rep18rep p

LG1(180)

2546/2008

Gr

BH

202(17/18)

3BHderivativ

eof

B2

transposon

on∼3

70-kb

plasmid

ofunknow

nrep-type,followed

bytransposition

ofBH

amongplasmidsor

plasmid

recombinatio

n

rep17rep18TA

1(30)

rep17rep18

TA1(155)

(370)

8744/2010

Wa-2

B2

561(17/18)

1rep2

rep17TA

2relpEF1

(35)

484/2010

Ke

B2

17(17/18)

3rep2

rep17rep18TA

1(<30)

rep2

(160)

rep2

rep17rep18

TA1(320)

Eur J Clin Microbiol Infect Dis (2017) 36:313–328 323

Tab

le2

(contin

ued)

StrainID

/year

ofisolation

Codeofmedicalcentrea

Tn1546

type

MLST

type

(lineage)

Num

berofV

anA

plasmid

bands

Hypotheticalgenetic

eventb

VanAplasmid

replicon

typesandstabilizatio

nsystem

s(approximatesize

inkb)c

Transferof

<30-kbplasmid

amongstrainsof

ST877

andST17

9363/2010

SwB2

877(17/18)

1rep2

rep17rep18

TA1(<30)

3856/2005

Wa-1

B4

78(78)

1rep2

TA2(50)

991/2009

eWa-4

G18(17/18)

1Recom

binatio

neventsor

transposition

ofG

amongrep17plasmids.

rep17(100)

3554/2009e

Wa-10

G192(78)

1rep17(50)

2944/2009

Ko

G18(17/18)

1rep2

rep17TA

2(35)

3392/2009

Ost

G78(78)

1rep17relpEF1

(45)

726/2010

InG

17(17/18)

1rep17relpEF1

(100)

3322/2007

Wa-2

BC1

412(78)

1Evolutio

nof

BCtransposons

with

inthesameST412

straininthe∼3

5-kb

plasmid

backbone

rep2

rep17TA

2(35)

107/2010

Wa-2

BC5

412(78)

1rep2

rep17TA

2(35)

3948/2010

Wa-2

BC4

412(78)

1rep2

rep17TA

2(35)

1901/2005

LoF

279(17/18)

1rep17rep18TA

2(50)

8034/2010

Kr-5

B3

341(78)

2rep2

rep17TA

1TA

2relpEF1

(50)

rep2

rep17TA

2relpEF1

(65)

8628/2010

Ka

BBI

202(17/18)

1(115)

ntnon-typeable

aByBydgoszcz,G

d-aGdańsk,adulth

emathology

ward;

Gd-pGdańsk,paediatrichaem

atologyward;

Gdy

Gdynia,GrGrodziskMazow

iecki,In

Inow

rocław

,KaKatow

ice,KeKętrzyn;K

oKonin,K

osKościerzyna,K

rKraków,LoŁó

dź,M

iMielec,OpOpole,O

sOstrówMazow

iecki,Osw

OstrówWielkopolski,OstOstrzeszów,O

tOtwock,PiP

isz,PlP

łock,P

oPo

znań,R

zRzeszów

,SkSkierniew

ice,Sw

Świdnica,W

aWarszaw

a,WrWrocław

,ZiZ

ielona

Góra;thecity

abbreviatio

nisfollo

wed

bythecentrenumberbSh

adow

edboxesindicatepresum

ableassociations

amongisolates;c

rep1,rep2,rep17,

rep18–plasmidreplicon

families

accordingtoJensen

etal.,2010

[24];T

A1,TA

2-axe-txeandω-ε-ζstabilizatio

nsystem

sspecificforpR

UM

andpR

E25,respectively;

dPresum

ablyC1transposon

type,

however

noam

plificationof

theregion

containing

IS1251

couldbe

obtained,inspite

ofseveralattempts

eResultsfrom

Wardaletal.,

2014

[18]

324 Eur J Clin Microbiol Infect Dis (2017) 36:313–328

earlier in orf2-vanR and vanS-vanR intergenic regions [40,41], as well as within IS1542 [42]. The F type harbouredISEfm2, representative of the IS256 family. Thus far, inenterococci this IS has been solely observed inserted be-tween orf13 and tetS within CTn6000 transposon [43]. Inour study, we report for the first time the insertion ofISEfm2 into Tn1546. The G type transposon with ISEf1,reported earlier for the VREfm outbreak in twoneighbouring Warsaw medical centres in 2009 [18] wasadditionally detected in isolates from 2009 to 2010 de-rived from seven different cities, which may indicate itsmulticenter spread. Integration of the ISEfm1-like element,belonging to the IS982 family in the BH type, represents yetanother novel insertion event in Tn1546. The presence ofISEfm1 was described previously in the vanX-vanY intergenicregion [44], as well as within the vanD operon [45]. Complexanalysis of transposon structures described in our studyrevealed the potential scheme of their hypothetical evolutionamong Polish VREfm. In this scenario, we propose the A1 typeas presumable ancestor variant, with remaining types being itsdirect or indirect derivatives (Fig. 3).

The vanA determinants are almost exclusively locatedon plasmids and these elements play a very important

role in the spread of glycopeptides resistance [46]. Ourresults show that the Polish VREfm population isenriched in plasmid replicons of different families in-cluding megaplasmids, Inc18-, pRUM- and pMG1/pHT-like plasmids, encountered in VanA-VREfm in oth-e r coun t r i e s [16 , 46 , 47 ] . S i ze va r i a t i on o fvanA-plasmids, even within the same family indicatestheir flexibility, and identification of multiple rep typesin a single plasmid suggests a common presence ofplasmid cointegrates. We observed an interesting changein the VanA-associated plasmidome between early(1997–2005) and more recent (2006–2010) isolates. Inparticular, rep1-vanA replicons were quite abundantamong early isolates and typically located on plasmidsover 140 bp in size. Together with rep2-vanA replicons theyrepresent the Inc18 family, associated with Tn1546 elementsamong clinical E. faecium in Europe [16, 47]. Another shift inplasmidome composition between early and recent isolateswas shown for pMG1 replicons, present exclusively amongearly VREfm. High prevalence of pMG1-like elements wasobserved among VREfm in the United States and Japan, wherethey contributed to the spread of both aminoglycoside andglycopeptide resistance [46]. Apart from Inc18 plasmids, the

A1

A2D

F

A5

G

B3

A3A6

A4

E

X1C1

BC2

C2

BB1,BB2B1

B2

BHB4

BBBI:1,2

X2BI

BBI

IS1216

IS1216

IS3-like

IS1251

IS1216

IS1216

ISEfm1-likeISEf1

BC:1,3,4,5

IS1216

Wa

Kr

Kr

Po

GdFig. 3 Hypothetical evolution of Tn1546 structures among PolishVREfm VanA. Type A1, found in different cities, is a presumableancestor variant with remaining types being its direct or indirectderivatives. Types A2, A3 and A5 developed by point mutations in wttype. A4 developed from A3 through single deletion events in the vanS-vanH intergenic region. A6 is an A3 derivative that lacks ca. 1900 bps inthe 5′ end. The E type transposon, a third potential derivative of A3, arosethrough acquisition of ISEfa4 between vanX and vanY. A3 and itsderivatives were typical for Poznań (Po) medical centres. B3 and G var-iants presumably developed from A5 after insertion events of IS1216 andISEf within vanX-vanY intergenic region in Kraków (Kr) and Warsaw(Wa), respectively. The ubiquitous B2 type, typical for Warsaw, probablyemerged from a single insertion event of IS1216within the A1 typewith a

concomitant deletion of the 5′ end of the transposon. The B4 (additionalsingle nucleotide insertion within vanY) and BH (ISEfm1-like insertionbetween vanX and vanY) types represent possible derivatives of B2. TheB1, D and F transposon types are potential derivatives of A1 formed byIS1216, ISEfa5 and ISEfm2 insertions, respectively. Another group oftransposon variants, encompassing types BI, BBI, BBBI1, BBBI2, BB1and BB2 emerged through complex insertion and deletion events in dif-ferent regions of wt transposon promoted mostly by IS1216 elements.This group was detected mainly in Gdańsk (Gd). The activity of anotherinsertion sequence, IS1251, followed by IS1216 insertions and severalpoint mutations resulted in the formation of C- and BC-types in Krakówand Warsaw, respectively

Eur J Clin Microbiol Infect Dis (2017) 36:313–328 325

pRUM derivatives constitute the second main carrier of van-comycin resistance among the contemporary E. faecium iso-lates [16, 46]. Plasmids with the pRUM-like rep can be dividedinto two groups, one with axe-txe genes and mob regions fromthe staphylococcal pC223 plasmid and the other with relaxasefrom pEF1 and lacking axe-txe [15, 16, 46]. Our results indi-cate that representatives of both these groups are presentamong Polish VREfm. Additionally, we observed plasmidswith unknown rep types among isolates obtained since 2006,which suggests the appearance of a new vanA-plasmid type(s),not included in the available classification scheme [24] andwhich will be a subject to further studies.

Finally, the analysis of PFGE-S1 hybridization resultsin the context of epidemiological information, determinedTn1546 types and the clonal background of the isolates,which revealed a high complexity of genetic events in-volving VREfm with VanA phenotype and resulting inthe dissemination of this type of resistance. As these 52isolates were pre-selected for a maximal representation ofthe collection diversity, only a few examples of clonalspread were observed, such as dissemination of ST132strain with a 45-kb plasmid harbouring C1 transposon ina Krakow hospital (Table 2). The role of VREfm clonalspread in Poland, however, had been demonstrated beforein our outbreak studies [18, 39, 48]. Particular types oftransposons in the analysed group were frequently associ-ated with various plasmid vectors. This situation mayhave resulted from transposition of Tn1546 among plas-mids [12], promoted by integration ‘hot-spots’ [49] andfrom the recombination processes among enterococcalplasmids [15, 50]. The present study also provided exam-ples of involvement of plasmids as vectors of vancomycinresistance, by demonstrating the presence of plasmids ofthe same size and with the same transposon types andplasmid-specific genes in different strains, as found inother studies [15, 51]. Occasionally, vanA-plasmids ap-peared to be integrated into bacterial chromosome, inagreement with other observations [52]. Further detailedstudies employing extensive sequencing are indispensableto fully elucidate the events involving genetic elementsengaged in the dissemination of the vanA gene cluster inthe population of Polish VREfm.

In conclusion, the VREfm of the VanA phenotype collectedin our country over the period 1997–2010 represent a highlyvariable group in the respect of their clonal composition, plas-mid content and structures of Tn1546, a direct carrier of vanAgenes. High genetic plasticity of these organisms, togetherwith a rapid global spread of successful hospital-adapted en-terococcal clones constitute a significant and continuouslyincreasing epidemiological threat for human health. Thus,both epidemiological situation concerning VREfm as well asgenetic elements and strains associated with VanA vancomy-cin resistance warrant further studies.

Acknowledgments We thank all Polish microbiologists who sentVREm isolates to our laboratory, Janetta Top for assigning new alleles,STs and MTs, and Kenneth Van Horn for critical reading of the manu-script. This publication made use of the Enterococcus faecium MLSTwebsite (http://efaecium.mlst.net/) hosted at Imperial College of theUniversity of Oxford and funded by the Wellcome Trust.

Compliance with ethical standards

Funding This work was supported by the grant N N401588540 fromthe Narodowe Centrum Nauki (NCN), Poland, by the MIKROBANKfunding from the Ministry of Science and Higher Education, Poland,and by a statutory funding from the Ministry of Science and HigherEducation, Poland.

Conflict of interest The authors declare that they have no conflict ofinterest.

Ethical approval and informed consent Isolates were obtained as apart of routine activity of the NRCSTand were analysed anonymously ina retrospective manner. Ethical approval and informed consent were thusnot required.

Open Access This article is distributed under the terms of the CreativeCommons At t r ibut ion 4 .0 In te rna t ional License (h t tp : / /creativecommons.org/licenses/by/4.0/), which permits unrestricted use,distribution, and reproduction in any medium, provided you giveappropriate credit to the original author(s) and the source, provide a linkto the Creative Commons license, and indicate if changes were made.

References

1. Hryniewicz W, Szczypa K, Bronk M, Samet A, Hellmann A,Trzcinski K (1999) First report of vancomycin-resistantEnterococcus faecium isolated in Poland. Clin Microbiol Infect5(8):503–505

2. Willems RJ, Top J, van Santen M, Robinson DA, Coque TM,Baquero F, Grundmann H, Bonten MJ (2005) Global spread ofvancomycin-resistant Enterococcus faecium from distinct nosoco-mial genetic complex. Emerg Infect Dis 11(6):821–828.doi:10.3201/eid1106.041204

3. Willems RJ, Hanage WP, Bessen DE, Feil EJ (2011) Populationbiology of gram-positive pathogens: high-risk clones for dissemi-nation of antibiotic resistance. FEMS Microbiol Rev 35(5):872–900. doi:10.1111/j.1574-6976.2011.00284.x

4. Willems RJ, Top J, van Schaik W, Leavis H, Bonten M, Siren J,Hanage WP, Corander J (2012) Restricted gene flow among hospi-tal subpopulations of Enterococcus faecium. MBio 3(4):e00151–00112. doi:10.1128/mBio.00151-12

5. Hendrickx AP, Bonten MJ, van Luit-Asbroek M, SchapendonkCM, Kragten AH, Willems RJ (2008) Expression of two distincttypes of pili by a hospital-acquired Enterococcus faecium isolate.Microbiology 154(Pt 10):3212–3223. doi:10.1099/mic.0.2008/020891-0

6. Heikens E, van Schaik W, Leavis HL, Bonten MJ, Willems RJ(2008) Identification of a novel genomic island specific tohospital-acquired clonal complex 17 enterococcus faecium isolates.Appl Environ Microbiol 74(22):7094–7097. doi:10.1128/AEM.01378-08

7. 7. Lebreton F, van Schaik W, McGuire AM, Godfrey P, Griggs A,Mazumdar V, Corander J, Cheng L, Saif S, Young S, Zeng Q,

326 Eur J Clin Microbiol Infect Dis (2017) 36:313–328

Wortman J, Birren B, Willems RJ, Earl AM, Gilmore MS (2013)Emergence of epidemic multidrug-resistant Enterococcus faeciumfrom animal and commensal strains. MBio 4(4). doi:10.1128/mBio.00534-13

8. Top J, Sinnige JC, Majoor EA, Bonten MJ,Willems RJ, van SchaikW (2011) The recombinase IntA is required for excision of esp-containing ICEEfm1 in Enterococcus faecium. J Bacteriol 193(4):1003–1006. doi:10.1128/JB.00952-10

9. Willems RJL, HomanW, Top J, van Santen-Verheuvel M, Tribe D,Manzioros X, Gaillard C, Vandenbroucke-Grauls CMJE, MasciniEM, van Kregten E, van Embden JDA, Bonten MJM (2001)Variant esp gene as a marker of a distinct genetic lineage ofvancomycinresistant Enterococcus faecium spreading in hospitals.Lancet 357(9259):853–855. doi:10.1016/s0140-6736(00)04205-7

10. Werner G, Fleige C, Geringer U, van Schaik W, Klare I, Witte W(2011) IS element IS16 as a molecular screening tool to identifyhospital-associated strains of Enterococcus faecium. BMC InfectDis 11:80. doi:10.1186/1471-2334-11-80

11. Courvalin P (2005) Genetics of glycopeptide resistance in gram-positive pathogens. Int J Med Microbiol 294(8):479–486.doi:10.1016/j.ijmm.2004.10.002

12. Arthur M, Molinas C, Depardieu F, Courvalin P (1993)Characterization of Tn1546, a Tn3-related transposon conferringglycopeptide resistance by synthesis of depsipeptide peptidoglycanprecursors in Enterococcus faecium BM4147. J Bacteriol 175(1):117–127

13. Talebi M, Pourshafie MR, Katouli M, Mollby R (2008) Molecularstructure and transferability of Tn1546-like elements inEnterococcus faecium isolates from clinical, sewage, and surfacewater samples in Iran. Appl Environ Microbiol 74(5):1350–1356.doi:10.1128/AEM.02254-07

14. Werner G, Klare I, Fleige C, Witte W (2008) Increasing rates ofvancomycin resistance among Enterococcus faecium isolated fromGerman hospitals between 2004 and 2006 are due to wide clonaldissemination of vancomycin-resistant enterococci and horizontalspread of vanA clusters. Int J Med Microbiol 298(5–6):515–527.doi:10.1016/j.ijmm.2007.05.008

15. Freitas AR, Novais C, Tedim AP, Francia MV, Baquero F, Peixe L,Coque TM (2013) Microevolutionary events involving narrow hostplasmids influences local fixation of vancomycin-resistance inEnterococcus populations. PLoS One 8(3):e60589. doi:10.1371/journal.pone.0060589

16. Rosvoll TC, Pedersen T, Sletvold H, Johnsen PJ, Sollid JE,Simonsen GS, Jensen LB, Nielsen KM, Sundsfjord A (2010)PCR-based plasmid typing in Enterococcus faecium strains revealswidely distributed pRE25-, pRUM-, pIP501- and pHTbeta-relatedreplicons associated with glycopeptide resistance and stabilizingtoxin-antitoxin systems. FEMS Immunol Med Microbiol 58(2):254–268. doi:10.1111/j.1574-695X.2009.00633.x

17. Sadowy E, Sienko A,Gawryszewska I, Bojarska A,MalinowskaK,Hryniewicz W (2013) High abundance and diversity of antimicro-bial resistance determinants among early vancomycin-resistantEnterococcus faecium in Poland. Eur J Clin Microbiol Infect Dis32(9):1193–1203. doi:10.1007/s10096-013-1868-y

18. Wardal E, Markowska K, Zabicka D, Wroblewska M, Giemza M,Mik E, Polowniak-Pracka H, Wozniak A, Hryniewicz W, SadowyE (2014) Molecular analysis of vanA outbreak of Enterococcusfaecium in twoWarsaw hospitals: the importance of mobile geneticelements. Biomed Res Int 2014:575367. doi:10.1155/2014/575367

19. CLSI (2015) Clinical and Laboratory Standards Institute (CLSI)(2015) Performance standards for antimicrobial susceptibility test-ing; 25th informational supplement. CLSI document M100-S20.CLSI, Wayne, PA

20. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME,Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-LiljequistB, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A,

Weber JT, Monnet DL (2012) Multidrug-resistant, extensivelydrug-resistant and pandrug-resistant bacteria: an international ex-pert proposal for interim standard definitions for acquired resis-tance. Clin Microbiol Infect 18(3):268–281. doi:10.1111/j.1469-0691.2011.03570.x

21. Clark NC, Cooksey RC, Hill BC, Swenson JM, Tenover FC (1993)Characterization of glycopeptide-resistant enterococci from U.S.hospitals. Antimicrob Agents Chemother 37(11):2311–2317

22. Top J, Schouls LM, Bonten MJ, Willems RJ (2004) Multiple-locusvariable-number tandem repeat analysis, a novel typing scheme tostudy the genetic relatedness and epidemiology of Enterococcusfaecium isolates. J Clin Microbiol 42(10):4503–4511. doi:10.1128/JCM.42.10.4503-4511.2004

23. HomanWL, Tribe D, Poznanski S, Li M, Hogg G, Spalburg E, VanEmbden JD, Willems RJ (2002) Multilocus sequence typingscheme for Enterococcus faecium. J Clin Microbiol 40(6):1963–1971

24. Jensen LB, Garcia-Migura L, Valenzuela AJ, Lohr M, Hasman H,Aarestrup FM (2010) A classification system for plasmids fromenterococci and other Gram-positive bacteria. J MicrobiolMethods 80(1):25–43. doi:10.1016/j.mimet.2009.10.012

25. Wardal E, Gawryszewska I, Hryniewicz W, Sadowy E (2013)Abundance and diversity of plasmid-associated genes among clin-ical isolates of Enterococcus faecalis. Plasmid 70(3):329–342.doi:10.1016/j.plasmid.2013.07.003

26. Barton BM, Harding GP, Zuccarelli AJ (1995) A general methodfor detecting and sizing large plasmids. Anal Biochem 226(2):235–240. doi:10.1006/abio.1995.1220

27. Willems RJ, Top J, van den Braak N, van Belkum A, Mevius DJ,Hendriks G, van Santen-Verheuvel M, van Embden JD (1999)Molecular diversity and evolutionary relationships of Tn1546-likeelements in enterococci from humans and animals. AntimicrobAgents Chemother 43(3):483–491

28. Jensen LB, Ahrens P, Dons L, Jones RN, Hammerum AM,Aarestrup FM (1998) Molecular analysis of Tn1546 inEnterococcus faecium isolated from animals and humans. J ClinMicrobiol 36(2):437–442

29. Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, RiceLB, Scheld M, Spellberg B, Bartlett J (2009) Bad bugs, no drugs:no ESKAPE! An update from the Infectious Diseases Society ofAmerica. Clin Infect Dis 48(1):1–12. doi:10.1086/595011

30. Werner G, Coque TM, Hammerum AM, Hope R, Hryniewicz W,Johnson A, Klare I, Kristinsson KG, Leclercq R, Lester CH, LillieM, Novais C, Olsson-Liljequist B, Peixe LV, Sadowy E, SimonsenGS, Top J, Vuopio-Varkila J, Willems RJ, Witte W, Woodford N(2008) Emergence and spread of vancomycin resistance amongenterococci in Europe. Euro Surveill 13(47)

31. Gawryszewska I, Zabicka D, Bojarska K, Malinowska K,Hryniewicz W, Sadowy E (2016) Invasive enterococcal infectionsin Poland: the current epidemiological situation. Eur J ClinMicrobiol Infect Dis 35(5):847–856. doi:10.1007/s10096-016-2607-y

32. Johnson PD, Ballard SA, Grabsch EA, Stinear TP, Seemann T,Young HL, Grayson ML, Howden BP (2010) A sustained hospitaloutbreak of vancomycin-resistantEnterococcus faecium bacteremiadue to emergence of vanB E. faecium sequence type 203. J InfectDis 202(8):1278–1286. doi:10.1086/656319

33. Zheng B, Tomita H, Xiao YH, Wang S, Li Y, Ike Y (2007)Molecular characterization of vancomycin-resistant enterococcusfaecium isolates from mainland China. J Clin Microbiol 45(9):2813–2818. doi:10.1128/JCM.00457-07

34. Khan MA, Northwood JB, Loor RG, Tholen AT, Riera E, FalconM, Paraguayan Antimicrobial N, van Belkum A, van WestreenenM, Hays JP (2010) High prevalence of ST-78 infection-associatedvancomycin-resistant Enterococcus faecium from hospitals in

Eur J Clin Microbiol Infect Dis (2017) 36:313–328 327

Asuncion, Paraguay. Clin Microbiol Infect 16(6):624–627.doi:10.1111/j.1469-0691.2009.02898.x

35. Arthur M, Depardieu F, Reynolds P, Courvalin P (1996)Quantitative analysis of the metabolism of soluble cytoplasmicpeptidoglycan precursors of glycopeptide-resistant enterococci.Mo l Mic rob io l 21 (1 ) : 33–44 . do i : 10 . 1046 / j . 1365 -2958.1996.00617.x

36. Khan SA, SungK, Layton S, NawazMS (2008) Heteroresistance tovancomycin and novel point mutations in Tn1546 of Enterococcusfaecium ATCC 51559. Int J Antimicrob Agents 31(1):27–36.doi:10.1016/j.ijantimicag.2007.08.007

37. Handwerger S, Skoble J (1995) Identification of chromosomal mo-bile element conferring high-level vancomycin resistance inEnterococcus faecium. Antimicrob Agents Chemother 39(11):2446–2453

38. Jung WK, Hong SK, Lim JY, Lim SK, Kwon NH, Kim JM, KooHC, Kim SH, Seo KS, Ike Y, Tanimoto K, Park YH (2006)Phenotypic and genetic characterization of vancomycin-resistantenterococci from hospitalized humans and from poultry in Korea.FEMS Microbiol Lett 260(2):193–200. doi:10.1111/j.1574-6968.2006.00311.x

39. Kawalec M, Kedzierska J, Gajda A, Sadowy E, Wegrzyn J, NaserS, Skotnicki AB, Gniadkowski M, Hryniewicz W (2007) Hospitaloutbreak of vancomycin-resistant enterococci caused by a singleclone of Enterococcus raffinosus and several clones ofEnterococcus faecium. Clin Microbiol Infect 13(9):893–901.doi:10.1111/j.1469-0691.2007.01774.x

40. Gu L, Cao B, Liu Y, Guo P, Song S, Li R, Dai H, Wang C (2009) Anew Tn1546 type of VanB phenotype-vanA genotype vancomycin-resistant Enterococcus faecium isolates in mainland China. DiagnMic rob io l I n f e c t D i s 63 (1 ) : 70–75 . do i : 10 . 1016 / j .diagmicrobio.2008.08.018

41. Depardieu F, Reynolds PE, Courvalin P (2003) VanD-type vanco-mycin-resistant Enterococcus faecium 10/96A. Antimicrob AgentsChemother 47(1):7–18. doi:10.1128/aac.47.1.7-18.2003

42. Cha JO, Yoo JI, Kim HK, Kim HS, Yoo JS, Lee YS, Jung YH(2013) Diversity of Tn1546 in vanA-positive Enterococcus faeciumclinical isolates with VanA, VanB, and VanD phenotypes and sus-ceptibility to vancomycin. J Appl Microbiol 115(4):969–976.doi:10.1111/jam.12300

43. Novais C, Freitas AR, Silveira E, Baquero F, Peixe L, Roberts AP,Coque TM (2012) Different genetic supports for the tet(S) gene inenterococci. Antimicrob Agents Chemother 56(11):6014–6018.doi:10.1128/AAC.00758-12

44. Novais C, Freitas AR, Sousa JC, Baquero F, Coque TM, Peixe LV(2008) Diversity of Tn1546 and its role in the dissemination ofvancomycin-resistant enterococci in Portugal. Antimicrob AgentsChemother 52(3):1001–1008. doi:10.1128/AAC.00999-07

45. Boyd DA, Conly J, Dedier H, Peters G, Robertson L, Slater E,Mulvey MR (2000) Molecular characterization of the vanD genecluster and a novel insertion element in a vancomycin-resistantEnterococcus isolated in Canada. J Clin Microbiol 38(6):2392–2394

46. Lanza VF, Tedim AP, Martinez JL, Baquero F, Coque TM (2015)The plasmidome of firmicutes: impact on the emergence and thespread of resistance to antimicrobials. Microbiol Spectr 3 (2):PLAS-0039-2014. doi:10.1128/microbiolspec.PLAS-0039-2014

47. Sletvold H, Johnsen PJ, Wikmark OG, Simonsen GS, SundsfjordA, Nielsen KM (2010) Tn1546 is part of a larger plasmid-encodedgenetic unit horizontally disseminated among clonal Enterococcusfaecium lineages. J Antimicrob Chemother 65(9):1894–1906.doi:10.1093/jac/dkq219

48. Kawalec M, Gniadkowski M, Hryniewicz W (2000) Outbreak ofvancomycin-resistant enterococci in a hospital in Gdask, Poland,due to horizontal transfer of different Tn1546-like transposon var-iants and clonal spread of several strains. J Clin Microbiol 38(9):3317–3322

49. Garcia-Migura L, Hasman H, Svendsen C, Jensen LB (2008)Relevance of hot spots in the evolution and transmission ofTn1546 in glycopeptide-resistant Enterococcus faecium (GREF)from broiler origin. J Antimicrob Chemother 62(4):681–687.doi:10.1093/jac/dkn265

50. Teuber M, Schwarz F, Perreten V (2003) Molecular structure andevolution of the conjugative multiresistance plasmid pRE25 ofEnterococcus faecalis isolated from a raw-fermented sausage. IntJ Food Microbiol 88(2–3):325–329

51. Garcia-Migura L, Liebana E, Jensen LB (2007) Transposon char-acterization of vancomycin-resistantEnterococcus faecium (VREF)and dissemination of resistance associated with transferable plas-mids. J Antimicrob Chemother 60(2):263–268. doi:10.1093/jac/dkm186

52. Paulsen IT, Banerjei L, Myers GS, Nelson KE, Seshadri R, ReadTD, Fouts DE, Eisen JA, Gill SR, Heidelberg JF, Tettelin H,Dodson RJ, Umayam L, Brinkac L, Beanan M, Daugherty S,DeBoy RT, Durkin S, Kolonay J, Madupu R, Nelson W,Vamathevan J, Tran B, Upton J, Hansen T, Shetty J, Khouri H,Utterback T, Radune D, Ketchum KA, Dougherty BA, Fraser CM(2003) Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis. Science 299(5615):2071–2074.doi:10.1126/science.1080613

53. EUCAST (2015) European Committee on AntimicrobialSusceptibility Testing (EUCAST) (2015) Breakpoint tables for in-terpretation of MICs and zone diameters. Version 5.0, EUCAST,Basel, Switzerland

328 Eur J Clin Microbiol Infect Dis (2017) 36:313–328